iNOS-GSNOR双通道控制机制在肠胶质细胞调控肠粘膜屏障功能中的作用研究

Intestinal epithelial barrier (IEB) plays a central role in maintaining internal hemostasis of the body. The key influence of enteric glial cells (EGC) in IEB regulation has been demonstrated in our previous study. EGC-derived inductible nitric oxide synthase (iNOS) plays complicated roles in IEB regulation under various conditions. Recent study suggested that there may exist iNOS- S-nitrosoglutathione reductase (GSNOR) double gate control in manipulating the synthesis and metabolism of NO and its related molecule S-nitrosoglutathione (GSNO), the latter demonstrating the barrier-protective properties. Our aim is to investigate the role of iNOS-GSNOR double gate control in the metabolism of NO-GSNO in EGCs through interfering the expression and activities of two key molecules, prove the influence of iNOS-GSNOR in the mechanisms of EGC in IEB regulation, and further clarify the exact role of EGC-derived iNOS. Meanwhile, through GSNO administration in in vitro and in vivo IEB experiments, the targeted proteins with S-nitrosylation in intestinal epithelial cells will be investigated with biotin-switch technique and other corresponding proteomics techniques. The goal of our study is to reveal the underlying mechanisms of EGC in IEB regulation under various pathophysiological conditions, and search potential drugs with IEB protective effects by the detection of protein S-nitrosylation.

肠粘膜屏障(IEB)是机体防御的重要屏障.前一课题里我们验证了肠胶质细胞(EGC)对IEB调控的关键作用,并发现生理及病理条件下诱导型一氧化氮合酶(iNOS)在EGC的IEB调控机制中扮演着不同角色.近来研究发现iNOS可能通过与亚硝基谷胱甘肽还原酶(GSNOR)协同形成双通道控制,来调节NO及其亚硝基化合物亚硝基谷胱甘肽(GSNO)合成与代谢,而GSNO为保护IEB的关键分子.本项目拟基于EGC-IEC共培养体系,通过干预EGC iNOS及GSNOR表达,对比观察生理及病理条件下EGC胞内NO-GSNO动态变化,验证iNOS-GSNOR双通道控制对EGC调控IEB的重要影响,进一步阐明EGC源iNOS在IEB调控中的作用;并以EGC源GSNO为切入点,利用体内外IEB损伤模型,通过Biotin-switch法及蛋白质组学技术,筛选IEB相关亚硝基化靶点蛋白,深入了解IEB调控分子机制.

肠粘膜屏障(IEB)是机体防御的重要屏障。在上一基金资助下我们发现诱导型一氧化氮合酶(iNOS)在 EGC的 IEB 调控机制中发挥重要作用，有研究发现iNOS 可能通过与亚硝基谷胱甘肽还原酶(GSNOR)协同调节一氧化氮(NO)及亚硝基谷胱甘肽(GSNO)合成与代谢。本项目基于EGC-IEC 共培养体系及小肠缺血再灌注（IR）小鼠模型，通过观察肠粘膜损伤程度、肠上皮通透性、跨上皮电阻、紧密连接（TJ）蛋白表达变化等指标，验证EGC源性GSNO在正常及IR、缺氧再复氧(HR) 等急性病理刺激下对肠粘膜屏障（IEB）功能的保护作用；通过干预EGC iNOS 及 GSNOR 表达,对比观察EGC 胞内 NO-GSNO动态变化，验证 iNOS-GSNOR 双通道控制机制对 EGC 调控IEB 的重要影响。本课题主要发现包括：（1）GSNO具有对IEB的保护作用并表现为剂量依赖性； GSNO预处理可明显缓解急性小肠IR刺激对小鼠肠粘膜的结构损伤，抑制肠粘膜通透性上升，肠上皮间TJ蛋白ZO-1及occludin表达明显上调；体外实验中GSNO预处理也可明显增强肠上皮细胞在正常及HR刺激下的IEB功能；（2）验证GSNOR是调节EGC细胞GSNO代谢的关键酶，HR及IR刺激诱导EGC内GSNOR过表达是EGC屏障功能受损的重要机制：western blot及免疫共定位研究均发现HR及IR刺激可诱导GSNOR表达明显上调，并伴随着EGC内GSNO含量明显下降； RNA干扰抑制EGC GSNOR表达，可显著抑制HR刺激诱导产生的EGC胞内GSNO含量下降，明显增强EGC的屏障保护功能；（3）发现了iNOS及GSNOR两个关键酶在EGC内GSNO代谢及 IEB调控功能的重要作用，初步验证了iNOS-GSNOR双通道机制在EGC屏障调控机制中的重要作用：iNOS抑制剂1400W预处理可显著增强EGC的IEB保护作用， 1400W可明显促进HR刺激导致的EGC胞内GSNO含量下调，而抑制GSNOR表达可显著缓解1400W所致的EGC GSNO含量下降，并增强EGC屏障保护功能。上述研究初步证实了iNOS-GSNOR 双通道控制机制是EGC GSNO代谢及IEB调控机制中的重要机制，为筛选新型临床IEB保护因子提供了重要依据。